[1] Department of Chemistry, University of Wisconsin, Madison, Wisconsin, USA. [2] These authors contributed equally to this work.
Nat Chem Biol. 2010 Apr;6(4):283-90. doi: 10.1038/nchembio.319. Epub 2010 Feb 28.
Specific interactions between post-translational modifications (PTMs) and chromatin-binding proteins are central to the idea of a 'histone code'. Here, we used a 5,000-member, PTM-randomized, combinatorial peptide library based on the N terminus of histone H3 to interrogate the multisite specificity of six chromatin binding modules, which read the methylation status of Lys4. We found that Thr3 phosphorylation, Arg2 methylation and Thr6 phosphorylation are critical additional PTMs that modulate the ability to recognize and bind histone H3. Notably, phosphorylation of Thr6 yielded the most varied effect on protein binding, suggesting an important regulatory mechanism for readers of the H3 tail. Mass spectrometry and antibody-based evidence indicate that this previously uncharacterized modification exists on native H3, and NMR analysis of ING2 revealed the structural basis for discrimination. These investigations reveal a continuum of binding affinities in which multisite PTM recognition involves both switch- and rheostat-like properties, yielding graded effects that depend on the inherent 'reader' specificity.
组蛋白翻译后修饰（PTMs）与染色质结合蛋白之间的特异性相互作用是“组蛋白密码”概念的核心。在这里，我们使用了一个由 5000 个成员组成的、基于组蛋白 H3 N 端的 PTM 随机组合肽文库，来探究六个染色质结合模块对赖氨酸 4 位甲基化状态的多位点特异性。我们发现，苏氨酸 3 位磷酸化、精氨酸 2 位甲基化和苏氨酸 6 位磷酸化是调节识别和结合组蛋白 H3 的能力的关键附加 PTM。值得注意的是，苏氨酸 6 位的磷酸化对蛋白质结合产生了最广泛的影响，这表明它是 H3 尾部阅读器的一个重要调控机制。质谱和基于抗体的证据表明，这种以前未被描述的修饰存在于天然 H3 上，对 ING2 的 NMR 分析揭示了区分的结构基础。这些研究揭示了一个连续的结合亲和力范围，其中多位点 PTM 识别既涉及开关样特性，又涉及变阻器样特性，从而产生依赖于固有“阅读器”特异性的分级效应。
